(‒)-Epicatechin reveals amoebicidal activity against Acanthamoeba castellanii by activating the programmed cell death pathway.

BACKGROUND: Acanthamoeba is an opportunistic pathogen that can cause human infections such as granulomatous amebic encephalitis and acanthamoeba keratitis. However, no specific drug to treat the diseases has been developed. Therefore, the discovery or development of novel drugs for treating Acanthamoeba infections is urgently needed. The anti-protozoan activity of (‒)-epicatechin (EC) has been reported, suggesting it is an attractive anti-protozoal drug candidate. In this study, the amoebicidal activity of EC against A. castellanii was assessed and its mechanism of action was unveiled. METHODS: The amoebicidal activity of EC against A. castellanii trophozoites and the cytotoxicity of EC in HCE-2 and C6 cells were determined with cell viability assay. The underlying amoebicidal mechanism of EC against A. castellanii was analyzed by the apoptosis/necrosis assay, TUNEL assay, mitochondrial dysfunction assay, caspase-3 assay, and quantitative reverse transcription polymerase chain reaction. The cysticidal activity of EC was also investigated. RESULTS: EC revealed amoebicidal activity against A. castellanii trophozoites with an IC50 of 37.01 ± 3.96 µM, but was not cytotoxic to HCE-2 or C6 cells. EC induced apoptotic events such as increases in DNA fragmentation and intracellular reactive oxygen species production in A. castellanii. EC also caused mitochondrial dysfunction in the amoebae, as evidenced by the loss of mitochondrial membrane potential and reductions in ATP production. Caspase-3 activity, autophagosome formation, and the expression levels of autophagy-related genes were also increased in EC-treated amoebae. EC led to the partial death of cysts and the inhibition of excystation. CONCLUSION: EC revealed promising amoebicidal activity against A. castellanii trophozoites via programmed cell death events. EC could be a candidate drug or supplemental compound for treating Acanthamoeba infections.

In vitro Evaluation the Efficacy of Some New Plant Extracts and Biocides on the Viability of Acanthamoeba castellanii.

The purpose of this study was to assess the efficacy of certain plant extracts and to compare them with current biocides on the viability of Acanthamoeba castellanii cysts and trophozoites in vitro. Amoebicidal and cysticidal assays were performed against both trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370). Ten plant extracts were evaluated alongside the current agents included polyhexamethylene biguanide (PHMB), octenidine and chlorhexidine digluconate. A. castellanii (ATCC 50370) was treated to serial two-fold dilutions of the test compounds and extracts in microtitre plate wells to investigate the effect on trophozoites and cysts of A. castellanii (ATCC 50370). Furthermore, the toxicity of each of the test compounds and extracts were assessed towards a mammalian cell line. Minimum trophozoite inhibitory concentration (MTIC), minimum trophozoite amoebicidal concentration (MTAC), and minimum cysticidal concentration (MCC) were used to establish A. castellanii (ATCC 50370) in vitro sensitivity. The findings of this research revealed that the biguanides PHMB, chlorhexidine, and octenidine all had excellent effectiveness against trophozoites and cysts of A. castellanii (ATCC 50370). The plant extracts testing results showed that, great activity against trophozoites and cysts ofA. castellanii (ATCC 50370) at lower concentrations. This is the first study to demonstrate that the Proskia plant extract had the lowest MCC value, which was 3.9 µg/mL. The time kill experiment confirmed this finding, as this extract reduced cysts of A. castellanii (ATCC 50370) by more than 3-log at 6 hour and by 4-log after 24 hour. The anti-amoebic efficacy of new plant extracts on the viability of A. castellanii (ATCC 50370) cysts and trophozoites was comparable to existing biocide treatments and was not toxic when tested on a mammalian cell line. This could be a promising novel Acanthamoeba treatment by using the tested plant extracts as a monotherapy against trophozoites and cysts.

The amoebicidal effect of Torreya nucifera extract on Acanthamoeba lugdunensis.

As the number of contact lens users increases, contact lens induced corneal infection is becoming more common. Acanthamoeba keratitis (AK) is a type of those which is caused by Acanthamoeba species, and may cause severe ocular inflammation and visual loss. We evaluated whether Torreya nucifera (T. nucifera) extract has an anti-amoebic effect and studied its mechanism of action on Acanthamoeba lugdunensis (A. lugdunensis). Cell viability was tested using the alamarBlue™ method, and the cell death mechanism was confirmed using the Tali® Apoptosis Kit. The SYTOX® Green assay was performed to check the plasma membrane permeability. The JC-1 dye was used to measure the mitochondrial membrane potential. A CellTiter-Glo® Luminescent Assay was used to measure the adenosine-triphosphate (ATP) level. Morphological changes in the mitochondria were examined by transmission electron microscopy (TEM). Cystic changes and a decrease in cell viability after treatment with T. nucifera were observed. Both apoptotic and necrotic cells were found in the Tali® Apoptosis assay. There was no significant difference in plasma membrane permeability between the control and T. nucifera treated groups. The collapse of the mitochondrial membrane potential and reduced ATP level in A. lugdunensis was confirmed in the groups treated with T. nucifera. Structural damage to the mitochondria was observed on TEM in the groups treated with T. nucifera. T. nucifera showed an anti-amoebic effect on A. lugdunensis, by inducing the loss of mitochondrial membrane potential. Thus, it could be a future therapeutic agent for AK.

Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level.

Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.

Acanthamoeba Keratitis: an update on amebicidal and cysticidal drug screening methodologies and potential treatment with azole drugs.

Introduction: Acanthamoeba encompasses several species of free-living ameba encountered commonly throughout the environment. Unfortunately, these species of ameba can cause opportunistic infections that result in Acanthamoeba keratitis, granulomatous amebic encephalitis, and occasionally systemic infection.Areas covered: This review discusses relevant literature found through PubMed and Google scholar published as of January 2021. The review summarizes current common Acanthamoeba keratitis treatments, drug discovery methodologies available for screening potential anti-Acanthamoeba compounds, and the anti-Acanthamoeba activity of various azole antifungal agents.Expert opinion: While several biguanide and diamidine antimicrobial agents are available to clinicians to effectively treat Acanthamoeba keratitis, no singular treatment can effectively treat every Acanthamoeba keratitis case.Efforts to identify new anti-Acanthamoeba agents include trophozoite cell viability assays, which are amenable to high-throughput screening. Cysticidal assays remain largely manual and would benefit from further automation development. Additionally, the existing literature on the effectiveness of various azole antifungal agents for treating Acanthamoeba keratitis is incomplete or contradictory, suggesting the need for a systematic review of all azoles against different pathogenic Acanthamoeba strains.

Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material.

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani. Additionally, a Microtox® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25-2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

PLGA nanoparticles loaded with Gallic acid- a constituent of Leea indica against Acanthamoeba triangularis.

Acanthamoeba, a genus that contains at least 24 species of free-living protozoa, is ubiquitous in nature. Successful treatment of Acanthamoeba infections is always very difficult and not always effective. More effective drugs must be developed, and medicinal plants may have a pivotal part in the future of drug discovery. Our research focused on investigating the in vitro anti- acanthamoebic potential of Leea indica and its constituent gallic acid in different concentrations. Water and butanol fractions exhibited significant amoebicidal activity against trophozoites and cysts. Gallic acid (100 µg/mL) revealed 83% inhibition of trophozoites and 69% inhibition of cysts. The butanol fraction induced apoptosis in trophozoites, which was observed using tunnel assay. The cytotoxicity of the fractions and gallic acid was investigated against MRC-5 and no adverse effects were observed. Gallic acid was successfully loaded within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with 82.86% encapsulation efficiency, while gallic acid showed 98.24% in vitro release at 48 hours. Moreover, the gallic acid encapsulated in the PLGA nanoparticles exhibited 90% inhibition against trophozoites. In addition, gallic acid encapsulated nanoparticles showed reduced cytotoxicity towards MRC-5 compared to gallic acid, which evidenced that natural product nanoencapsulation in polymeric nanoparticles could play an important role in the delivery of natural products.

Phytochemical Screening and Acanthamoebic Activity of Shoots from in Vitro Cultures and in Vivo Plants of Eryngium alpinum L.-The Endangered and Protected Species.

Genetically uniform shoots of Eryngium alpinum L. cultured in vitro were subjected to the qualitative analysis applying the UPLC-HESI-HRMS technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. The phytochemical screening of the crude methanolic extracts of shoots, both from in vitro cultures and in vivo plants, revealed the presence of phenolic acids, coumarins, flavonoids, triterpenoid saponins, amino acids, or dipeptides. Active compounds detected are known to have medicinal importance, and for this reason, the present study represents a preliminary investigation of the extracts against pathogenic and opportunistic amoeba. Among the extracts tested, the extract of shoots from in vitro cultures exhibited remarkable amoebicidal action against trophozoites. On the second day of treatment, the extract at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL showed the highest antiamoebicidal effect: the inhibition of trophozoites reached 81.14%, 66.38%, and 54.99%, respectively. To our best knowledge, the present report is the first to show the phytochemical screening and to discuss the antiamoebic activity of Eryngium alpinum L. shoots, both from in vitro cultures and in vivo plants.

In Vitro Screening of the Open-Source Medicines for Malaria Venture Malaria and Pathogen Boxes To Discover Novel Compounds with Activity against Balamuthia mandrillaris.

Balamuthia mandrillaris is an under-reported, pathogenic free-living amoeba that causes Balamuthia amoebic encephalitis (BAE) and cutaneous skin infections. Although cutaneous infections are not typically lethal, BAE with or without cutaneous involvement is usually fatal. This is due to the lack of drugs that are both efficacious and can cross the blood-brain barrier. We aimed to discover new leads for drug discovery by screening the open-source Medicines for Malaria Venture (MMV) Malaria Box and MMV Pathogen Box, with 800 compounds total. From an initial single point screen at 1 and 10 µM, we identified 54 hits that significantly inhibited the growth of B. mandrillarisin vitro Hits were reconfirmed in quantitative dose-response assays and 23 compounds (42.6%) were confirmed with activity greater than miltefosine, the current standard of care.

Evaluation of Oxasqualenoids from the Red Alga Laurencia viridis against Acanthamoeba.

Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.

Development of a Novel Ex-vivo 3D Model to Screen Amoebicidal Activity on Infected Tissue.

Amoebiasis is a parasitic disease that causes thousands of deaths every year, its adverse effects and resistance to conventional treatments have led to the search of new treatment options, as well as the development of novel screening methods. In this work, we implemented a 3D model of intestine and liver slices from hamsters that were infected ex vivo with virulent E. histolytica trophozoites. Results show preserved histology in both uninfected tissues as well as ulcerations, destruction of the epithelial cells, and inflammatory reaction in intestine slices and formation of micro abscesses, and the presence of amoebae in the sinusoidal spaces and in the interior of central veins in liver slices. The three chemically synthetized compounds T-001, T-011, and T-016, which act as amoebicides in vitro, were active in both infected tissues, as they decreased the number of trophozoites, and provoked death by disintegration of the amoeba, similar to metronidazole. However, compound T-011 induced signs of cytotoxicity to liver slices. Our results suggest that ex vivo cultures of precision-cut intestinal and liver slices represent a reliable 3D approach to evaluate novel amoebicidal compounds, and to simultaneously detect their toxicity, while reducing the number of experimental animals commonly required by other model systems.

Recombinant Buckwheat Trypsin Inhibitor Improves the Protein and Mitochondria Homeostasis in Caenorhabditis elegans Model of Aging and Age-Related Disease.

BACKGROUND: With the acceleration of aging process in human society, improvements of the physical functionality and life quality in the elderly population are more meaningful than pure longevity. Buckwheat trypsin inhibitor is a low molecular weight polypeptide extracted from buckwheat, which is a beneficial food for improving the health in the elderly. OBJECTIVES: The aim of the current study was to evaluate the potential beneficial effects of recombinant buckwheat trypsin inhibitor (rBTI) on age-dependent function decline and the primary mechanism. METHOD: Day 10 N2 Caenorhabditis elegans and day 6 AM140 C. elegans cultured at 25°C were used as models of aging and age-related disease, respectively. Motor function was as an indicator of age-dependent function. ATP content and damage mitochondrial DNA mass were detected to assess mitochondrial damage and function by ATP Assay Kit and agarose gel electrophoresis, respectively. Soluble protein content was quantified by SDS polyacrylamide gel electrophoresis. Autophagy-related genes transcription levels, autophagy marker proteins lgg-1, and lysosomal content were analyzed to quantify autophagy levels by qRT-PCR, transgenic C. elegans, and lysosomal staining. Autophagy inhibitor chloroquine, daf-16 mutant, and RNA Interference were used to determine the roles of autophagy and DAF-16 in rBTI-mediated effects. RESULTS: In this study, we found that rBTI could decrease the proportions of insoluble protein and impaired mitochondria, finally reduce motility deficits in both models. Further study indicated that rBTI activated the autophagy, and the inhibition of autophagy reduced rBTI-mediated beneficial effects. Genetic analyses showed the transcriptional activity of DAF-16 was increased by rBTI and was required for rBTI-mediated beneficial effects. CONCLUSIONS: These data indicated that rBTI might promote the autophagy to alleviate the age-related functional decline via DAF-16 in C. elegans and suggested a potential role of rBTI as a nutraceutical for the improvement of age-related complications.

Amoebicidal and Amoebistatic Effects of Artemisia argyi Methanolic Extracts on Acanthamoeba castellanii Trophozoites and Cysts.

PURPOSE : The present study aimed to investigate the amoebicidal and amoebistatic efects of Artemisia argyi leaf methanolic extract by testing the effects on trophozoites and on cysts. We also determined cytotoxic effect, enzymatic and non-enzymatic antioxidant activities, total phenolic, lavonoid and antioxidative contents of A. argyi. METHODS: A. argyi was harvested from various geographic sites in Ordu province in Turkey. The fresh leaves were subjected to methanolic extraction. In 100 µl culture, different concentrations of A. argyi methanolic extract (in quantities from 1.2, 2.3, 4.7, 9.4, 18.7, 37.4, 74.8 mg/ml) and the same volume of trophozoite/cyst suspension were mixed for the determination of the amoebicidal activity of the plant extract. Human bronchial epithelial cells were treated with the same concentrations of Artemisia extracts to determine cytotoxic potential. RESULTS: Total phenolic and lavonoid contents of the extract were calculated as 261 mg gallic acid/g dry extract and 29 mg quercetin/g dry extract, respectively. Total antioxidant activity was also calculated as 367 mg ascorbic acid/g dry extract. The growth of trophozoites stopped in A. argyi methanolic extract with 50% inhibitory concentrations (IC50)/8 h for 37.4 mg/ ml and 74.8 mg/ml extract solution and had stronger amoebicidal activity on the cysts with IC50/72 h. Artemisia showed stronger inhibitory effects on bronchial epithelial cells at the concentrations of 9.4, 18.7, 37.4 and 74.8 mg/ml. CONCLUSION: The study indicated that A. argyi leaf extract has cytotoxic and anti-amoebic activities.

Dual 0.02% chlorhexidine digluconate - 0.1% disodium EDTA loaded thermosensitive ocular gel for Acanthamoeba keratitis treatment.

Poor bioavailability and low residence time limit the efficiency of conventional biguanide-based eye drops against Acanthamoeba keratitis. The aim of this work was to formulate an original anti-amoebic thermoreversible ocular gel combining biguanide and metalloproteases inhibitor - chelating agent. Chlorhexidine digluconate (CHX)-ethylenediaminetetraacetic acid disodium salt (Na2EDTA) were compounded in poloxamer 407 saline solution. 0.02% CHX - 0.1% Na2EDTA loaded thermosensitive ocular gel exhibited appropriate pH (5.73 ±â€¯0.06), iso-osmolality (314 ±â€¯5 mOsm/kg), viscosity (ranged between 15 and 25 mPa.s) and thermal gelation (26.5 °C and 33 °C) properties. Bioadhesion of gel was successfully tested onto isolated bovine eyes as well as the assessment of CHX penetration into the cornea. Intracorneal CHX concentration was found greater than trophozoite minimum amoebicidal concentration and minimal cysticidal concentration after 15-min and 2-h ocular exposure, respectively, while any CHX permeation through the cornea was detected (<51 ng/cm2/h). Improvement of CHX ocular bioavailability was attributed to probable solubilization of tear film lipid layer by poloxamer. In vitro efficiency of CHX-Na2EDTA ocular gel was confirmed from the drastic reduction of trophozoite and cyst survival (to 25% and 2%, respectively), confirming the potential of the multicomponent pharmaceutical material strategy for the treatment of Acanthamoeba keratitis.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Correlation of radical-scavenging capacity and amoebicidal activity of Matricaria recutita L. (Asteraceae).

Some Acanthamoeba strains are able to cause Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide because of their pathogenicity. The treatment of Acanthamoeba infections is complicated due to the existence of a highly resistant cyst stage in their life cycle. Therefore, the elucidation of novel sources of anti-Acanthamoeba agents is an urgent need. In the present study, an evaluation of the antioxidant and anti-Acanthamoeba activity of compounds in flower extracts of Tunisian chamomile (Matricaria recutita L.) was carried out. Chamomile methanol extract was the most active showing an IC50 of 66.235 ± 0.390 µg/ml, low toxicity levels when checked in murine macrophage toxicity model and presented also antioxidant properties. Moreover, a bio-guided fractionation of this extract was developed and led to the identification of a mixture of coumarins as the most active fraction. These results suggest a novel source of anti-Acanthamoeba compounds for the development of novel therapeutic agents against Acanthamoeba infections.

Amoebicidal, antimicrobial and in vitro ROS scavenging activities of Tunisian Rubus ulmifolius Schott, methanolic extract.

The present study aimed to evaluate the activity of methanolic extract of Rubus ulmifolius Schott against the Acanthamoeba castellani Neff Strain as well as its antioxidant and antimicrobial effects. The tested extract has a good amoebicidal activity with low IC50 (61.785 ± 1.322 µg/ml) and also has significant activity against both Gram-positive (S. aureus, S. agalactiae) and Gram-negative bacteria (E. coli, S. typhimurium) and against C. albicans. The inhibition zones diameters (IZD) and minimal inhibitory concentration (MIC) values were in the range of 22.5-50 mm and 02.29-4.76 mg ml-1, respectively. In the other hand, the in vitro ROS scavenging activity was evaluated, the tested extract exhibited a good effect on the ·OH radical (89.99% at a concentration of 100 µg/ml) when compared to the ascorbic acid (68.81%). Moreover, the inhibition percentage of superoxide generation by R. ulmifolius extract at 100 µg/ml was greater than ascorbic acid (79.55; 64.79%, respectively). Also, the tested extract showed a high percentage of H2O2 scavenging activity (99.95% at 100 µg/ml). Our findings suggest that R. ulmifolius could be a potential source of natural antioxidant in preventing many diseases associated with oxidative stress, amoebic and bacterial infections.

The effect of viroid infection of citrus trees on the amoebicidal activity of 'Maltese half-blood' (Citrus sinensis) against trophozoite stage of Acanthamoeba castellanii Neff.

In order to promote a local Tunisian product, this study was designed to examine, for the first time, the anti-Acanthamoeba activity (Acanthamoeba castellanii Neff) of the essential oils of Tunisian Citrus sinensis peels (Maltese half-blood) and the effect of viroid plant infection on this activity. To do so, three samples of peels' essential oils were studied: from a healthy plant (Control), a plant inoculated with Citrus exocortis viroid (CEVd) and one inoculated with hot stunt cachexia viroid (HSVd). The samples were extracted by hydrodistillation from dried peels and characterized by GC-MS. Limonene was the major component with a percentage ranging from 90.76 to 93.34% for (CEVd) sample and (Control), respectively. Anti-Acanthamoeba activity of the tested oils was determined by the Alamar Blue® assay. Primary results showed a strong potential anti-Acanthamoeba activity with an IC50 ranging from 36.6 to 54.58 µg/ml for (HSVd) and (CEVd) samples, respectively. In terms of the effect of viroid infection, a strong positive correlation was observed between different chemical classes and anti-Acanthamoeba activity.

THE USE OF EXTRACTS FROM PASSIFLORA SPP. IN HELPING THE TREATMENT OF ACANTHAMOEBIASIS.

Chronic progressive diseases of the central nervous system such as granulomatous amoebic encephalitis, amoebic keratitis, amoebic pneumonitis and also skin infections caused by free-living amoebae (Acanhamoeba spp.) are a significant challenge for pharmacotherapy. This is due to the lack of effective treatment because of encystation, which makes the amoebae highly resistant to anti-amoebic drugs. A very inter- esting and promising source of future drugs in this area are plant materials obtained not only from the habitat but also from plant in vitro culture as an alternative source of biomaterials. Alcoholic extracts from leaves of Passiflora incarnata, P. caerulea, P. alata (Passifloraceae) and from callus cultures were evaluated in vito for amoebicidal activity. Phytochemical analysis showed that all extracts contained phenolic compounds including flavonoids? Biological study revealed that all extracts showed amoebostatic and amoebicidal properties in concentrations from 4 to 12 mg/mL. Extracts of P. alata leaf and callus showed the most effective activities (IC5, 4.01 mg/mL, IC,5 7.29 mg/mL, respectively) after 48 h of exposure, which was correlated with the highest concentration of total phenolics and flavonoids in comparison with other extracts.